This invention relates generally to mass spectrometers and to methods of performing mass spectroscopy. In particular, this invention relates to tandem time-of-flight mass spectrometers and to methods of performing mass spectroscopy using tandem time-of-flight mass spectrometers.
Mass spectrometers vaporize and ionize a sample of interest and determine the mass-to-charge ratio of the resulting ions. Time-of-flight (TOF) mass spectrometers determine the mass-to-charge ratio of an ion by measuring the amount of time it takes a given ion to migrate from an ion source to a detector, under the influence of electric fields. The time it takes for an ion to reach the detector, for electric fields of given field strengths, is a direct function of the ion""s mass and an inverse function of the ion""s charge.
Recently, TOF mass spectrometers have become widely accepted, particularly for the analysis of relatively nonvolatile biomolecules, and for other applications requiring high speed, high sensitivity, and/or wide mass range. New ionization techniques such as matrix-assisted laser desorption/ionization (MALDI) and electrospray (ESI) have greatly extended the mass range of molecules that can be analyzed by mass spectrometers. These techniques can produce intact molecular ions in a gas phase suitable for analysis.
TOF mass spectrometers have unique advantages for these applications. The recent development of delayed ion extraction, for example, as described in U.S. Pat. Nos. 5,625,184, 5,627,369, and 6,057,543 has made high resolution and precise mass measurement routinely available with MALDI-TOF mass spectrometers. The entire disclosures of U.S. Pat. Nos. 5,625,184, 5,627,369, and 6,057,543 are incorporated herein by reference. Orthogonal injection with pulsed extraction has provided similar performance enhancements for ESI-TOF. These techniques provide accurate data on the molecular weight of samples. However, these techniques provide little information on molecular structure.
Some prior art MALDI-TOF mass spectrometers use a technique known as post-source decay (PSD) to fragment the ions. However, the fragmentation spectra produced by PSD are often relatively weak and difficult to interpret. Other prior art MALDI-TOF mass spectrometers include a collision cell that causes some of the ions to undergo high energy collisions with neutral gas molecules to enhance the production of low mass fragment ions and to produce some additional fragmentation. However, these prior art mass spectrometers are not useful for every application.
Other prior art techniques, such as ion traps and Fourier-transform ion-cyclotron-resonance mass spectrometry (FT-ICR-MS), allow multiple steps of fragmentation of primary ions to be observed. These techniques provide a more detailed picture of the fragmentation and in some cases may allow more structural information to be obtained. However, these devices are limited to low energy collisional processes that do not provide some of the specificity provided by high energy collisional dissociation.
Still other prior art mass spectrometers use ESI-TOF that produce fragmentation by causing energetic collisions to occur in the interface between the atmospheric pressure electrospray and the evacuated mass spectrometer. However, these prior art mass spectrometers have no means for selecting a particular primary ion.
There are several prior art tandem mass spectrometers that are generally referred to as MS-MS instruments. MS-MS instruments use mass spectrometer techniques for selecting a primary ion and/or detecting and analyzing fragment ions. The most common form of tandem mass spectrometry is the triple quadrupole mass spectrometer. The first quadrupole selects the primary ion. The second quadrupole is typically maintained at a sufficiently high pressure and voltage so that multiple low energy collisions occur causing some of the ions to fragment. The third quadrupole is scanned to analyze the fragment ion spectrum. The resulting spectra are typically easy to interpret and numerous analysis techniques have been developed. For example, techniques have been developed for determining the amino acid sequence of a peptide from such spectra.
Another prior art tandem mass spectrometer uses two quadrupole mass filters and a TOF mass spectrometer. The first quadrupole selects the primary ion. The second quadrupole is maintained at a sufficiently high pressure and voltage so that multiple low energy collisions occur causing some of the ions to fragment. The TOF mass spectrometer detects and analyzes the fragment ion spectrum.
U.S. Pat. No. 5,202,563 describes a tandem time-of-flight mass spectrometer that includes a grounded vacuum housing, two reflecting-type mass analyzers coupled via a fragmentation chamber, and flight channels electrically floated with respect to the grounded vacuum housing. These mass spectrometers are generally limited to analyzing relatively small molecules and do not provide the sensitivity and resolution required for biological applications, such as sequencing of peptides or oligonucleotides.
For peptide sequencing and structure determination by tandem mass spectrometry, both mass analyzers must have adequate mass resolution and good ion transmission over the mass range of interest. MS-MS systems are typically used for peptide sequencing above a molecular weight of 1000. These systems may include two double-focusing magnetic deflection mass spectrometers having high mass range. Although these instruments provide high mass range and mass accuracy, they are limited in sensitivity, compared to time-of-flight mass spectrometers, and are not readily adaptable for use with modern ionization techniques, such as MALDI and electrospray. These instruments are also very complex and expensive.
Another prior art tandem mass spectrometer that uses time-of-flight mass spectrometer techniques includes two linear time-of-flight mass analyzers that use surface-induced dissociation (SID). One such mass spectrometer includes an ion mirror.
U.S. Pat. No. 5,206,508 describes a tandem mass spectrometer that uses either linear or reflecting analyzers, which are capable of obtaining tandem mass spectra for each parent ion without requiring the separation of parent ions of differing mass from each other.
Tandem mass spectrometers (MS-MS) employing time-of-flight can provide structural information. Such a tandem MS-MS instrument is described in U.S. Pat. No. 6,348,688, the entire disclosure of which is incorporated herein by reference. In this MS-MS instrument, a first mass analyzer is used to select a primary ion of interest, for example, a molecular ion of a particular sample. The ion of interest is then fragmented by increasing the internal energy of the ion. For example, the ion of interest can be fragmented by causing a collision of the ion with a neutral gas molecule. The mass spectrum of the fragment ions is then analyzed by a second mass analyzer. The structure of the primary ion can be determined by interpreting its fragmentation pattern.
The present invention relates to improving the performance of mass spectrometers. In one embodiment, a mass spectrometer according to the present invention includes a plurality of TOF mass separators operating in series in a TOF mass spectrometer. A mass separator of the present invention can separate and fragment ionic species generated by a previous mass separator, thereby providing increasingly detailed analysis of a chemical sample with each successive stage. One aspect of the mass spectrometer of the present invention is that modes of operation of the stages of mass spectrometric measurement can be selected electrically.
Accordingly, a tandem time-of-flight mass spectrometer (TOF-MS) of the present invention includes a pulsed ion source that generates a plurality of ions. In one embodiment, the pulsed ion source includes an injector that injects ions into a first field-free region, and a pulsed ion accelerator that extracts the plurality ions from the injected ions by accelerating the ions in a direction that is orthogonal to the direction of injection. In another embodiment, the pulsed ion source is a laser desorption/ionization ion source. In one embodiment, the pulsed ion source is a delayed extraction ion source that extracts the ions after a time delay following ionization. In one embodiment, the pulsed ion source is a pneumatically-assisted electrospray, chemical ionization, or ICP ion source.
The tandem TOF-MS of the present invention also includes a first, a second, and a third TOF mass separator positioned along a path of the plurality of ions generated by the pulsed ion source. The first mass separator is positioned to receive the plurality of ions generated by the pulsed ion source. The first mass separator accelerates the plurality of ions generated by the pulsed ion source, separates the plurality of ions according to their mass-to-charge ratio, and selects a first group of ions based on their mass-to-charge ratio from the plurality of ions. The first mass separator also fragments at least a portion of the first group of ions.
The second mass separator is positioned to receive the first group of ions and fragments thereof generated by the first mass separator. The second mass separator accelerates the first group of ions and fragments thereof, separates the first group of ions and fragments thereof according to their mass-to-charge ratio, and selects from the first group of ions and fragments thereof a second group of ions based on their mass-to-charge ratio. The second mass separator also fragments at least a portion of the second group of ions. The first and/or the second mass separator may also include an ion guide, an ion-focusing element, and/or an ion-steering element.
The third mass separator is positioned to receive the second group of ions and fragments thereof generated by the second mass separator. The third mass separator accelerates the second group of ions and fragments thereof and separates the second group of ions and fragments thereof according to their mass-to-charge ratio. In one embodiment, the third mass separator accelerates the second group of ions and fragments thereof using pulsed acceleration.
The tandem TOF-MS also includes an ion detector positioned to receive the second group of ions and fragments thereof. In one embodiment, the tandem TOF MS also includes an ion reflector positioned in a field-free region. The ion reflector corrects the energy of at least one of the first or second group of ions and fragments thereof before they reach the ion detector. In one embodiment, the tandem TOF-MS may also include a processor that determines the mass-to-charge ratio of ions detected by the ion detector. In one embodiment, the processor includes data processing equipment such as an embedded microprocessor or a stand-alone computer.
A tandem TOF-MS of the present invention can be configured in a variety of ways. In one embodiment, the second TOF mass separator accelerates the first group of ions and fragments thereof with a negative acceleration. Negative acceleration is also called deceleration. In one embodiment, the first TOF mass separator includes in a field-free region an ion selector that selects ions having a mass-to-charge ratio that is substantially within a first predetermined range.
In one embodiment, the second TOF mass separator includes a field-free region and an ion selector that selects ions having a mass-to-charge ratio that is substantially within a second predetermined range. In one embodiment at least one of the first and the second TOF mass separator includes a timed-ion-selector that selects fragmented ions.
In one embodiment, at least one of the first and the second mass separator includes an ion fragmentor. Numerous types of ion fragmentors are known in the art. For example, in one embodiment, the ion fragmentor includes a collision cell in which ions are fragmented by causing them to collide with neutral gas molecules. In another embodiment, the ion fragmentor includes a photodissociation cell that fragments ions by irradiating them with a beam of photons. In yet another embodiment, the ion fragmentor includes a surface dissociation fragmentor that fragments ions by colliding them with a solid or a liquid surface.
The present invention also features a method for high-resolution TOF mass spectrometry of fragmented ions that provides increased structural information. The method includes generating a pulse of ions from a sample of interest. In one embodiment, the pulse of ions is generated by using a method including one of electrospray, pneumatically-assisted electrospray, chemical ionizing, MALDI, and ICP.
Precursor ions are then selected from the pulse of ions during a time interval to form selected precursor ions, where the selected precursor ions have predetermined mass-to-charge ratios. In one embodiment, the precursor ions are selected by transmitting the selected precursor ions through a timed ion selector and by substantially blocking all other ions. The selected precursor ions are then fragmented. In one embodiment, the selected precursor ions are fragmented by colliding the selected precursor ions with neutral gas molecules, thereby exciting the selected precursor ions. In one embodiment, the selected precursor ions are fragmented by passing the selected precursor ions through a nearly field-free region, thereby allowing the selected precursor ions to substantially complete fragmentation.
Primary ion fragments are then selected from the fragmented selected precursor ions during a time interval to form selected primary ion fragments. In one embodiment, the kinetic energy of the selected primary ion fragments is adjusted. The selected primary ion fragments are then fragmented to form secondary ion fragments. In one embodiment, the selected primary ion fragments are passed through a nearly field-free region, thereby allowing the selected primary ion fragments to substantially complete fragmentation.
The secondary ion fragments are then separated in time from the selected primary ion fragments. In one embodiment, the secondary ion fragments are focused. At least one of the selected primary and the secondary ion fragments are detected as a function of time to produce a mass spectrum.
The method may also include adjusting the kinetic energy of the selected primary ion fragments. In one embodiment, the energy of the primary ion fragments is adjusted to compensate for changes in the mode of operation of a tandem TOF MS according to the present invention. In one embodiment, the method includes focusing the secondary ion fragments.